In addition to existing neuroblastoma (Cl300) and skeletal muscle myoblast cell lines, new cell lines of rat nerve, smooth and skeletal muscle, and glia have been established in clonal cell culture and will be characterized with respect to their physiological and biochemical properties. Attempts will also be made to isolate large numbers of cell lines from specific areas of the brain by techniques which have proven successful with the whole brain. Some of the five new neuronal cell lines synthesize gamma-aminobutyric acid and will be utilized to study the synthesis, uptake, storage, and release of this putative neurotransmitter; the other neuronal cell lines will be used for similar studies once their transmitters are defined. The process of neuronal and glial differentiation will be defined and compared to the Cl300 neuroblastoma. Similarly, the physiology, pharmacology, and biochemical differentiation of newly established smooth muscle cell lines will be examined. Since secreted material is likely to play a role in gradients and trophic effects between cells during morphogenesis, the secreted proteins and peptides of the nerve, glial, and muscle cell lines will be examined in detail. The role of glial and nerve antigenic markers will also be examined in relation to demyelinating diseases such as experimental allergic encephalomyelitis and multiple sclerosis. Finally, the trophic interaction between nerve and nerve, and nerve and muscle, will be examined with the intent of establishing a clonal system where the chemistry and physiology of synaptic specificity can be studied in detail.